Method for neutralization of antibiotics in a culture medium

ABSTRACT

The present invention is directed to a method and means for the neutralization, binding, and/or inactivation of antimicrobials in a test sample. The invention is also directed to a method of detecting the presence of one or more microorganisms in a test sample by culturing the test sample in a culture media comprising one or more primary amine-containing compounds.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 61/269,953, entitled, “Method for Neutralization ofAntibiotics in a Culture Medium”, filed Jul. 1, 2009, which isincorporated herein.

FIELD OF THE INVENTION

The present invention is directed to the neutralization and/orinactivation of antibiotics in a test sample or culture medium. Thepresent invention is also directed to a method of culturing anddetecting microorganisms that may be present in a test sample.

BACKGROUND OF THE INVENTION

In the field of culturing and detecting microorganisms, specializedculture bottles and machines for holding the culture bottles aretypically used for detecting the presence of microorganisms in a testspecimen. Bottles, such as those disclosed in U.S. Pat. Nos. 4,945,060;5,094,955; and 5,162,229, herein incorporated by reference, have aculture medium and a sensor in the interior of the bottle that undergoesa detectable change due to the growth of microorganisms present in thebottle. The change in the sensor is monitored from outside the culturebottle through the transparent wall of the culture bottle, such as witha light emitter and detector as disclosed in, for example, U.S. Pat.Nos. 5,164,796 and 5,217,876, herein incorporated by reference. For mostassays, the culture bottles should be agitated for best results. Clips,such as those disclosed in U.S. Pat. No. 5,074,505, herein incorporatedby reference, can hold the culture bottles in place in the incubatingmachine during agitation.

The detection of pathogenic microorganisms in biological fluids shouldbe performed in the shortest possible time, in particular in the case ofsepticemia for which the mortality remains high in spite of the broadrange of antibiotics which are available to doctors. In order toincrease sick individuals' chances of survival, practitioners oftenadminister an antibiotic or mixture of antibiotics to the patients. Itis, however, important to determine a suitable antibiotic therapy assoon as possible. Unfortunately, when a patient's bodily fluid sample iscultured to identify and isolate an infecting microorganism that mightbe present, antibiotics previously administered to the patient caninterfere with the culturing process. Furthermore, medical samples maycontain serum, plasma and lysed erythrocytes that may naturally containmicrobial inhibitors. Industrial samples such as pharmaceuticals andfoods may also contain antimicrobials or preservatives that inhibit thegrowth of microorganisms. Additionally, when culture media is prepared,autoclaving of the media at very high temperatures under pressure canresult in the formation of by-products toxic to microorganisms. Removalor neutralization of these inhibitory or bactericidal substances isnecessary to detect microbial contamination.

The use of resins and non-resinous adsorbents is well known and has beenpreviously described for use in medical diagnostic procedures. Inparticular, these resins and non-resinous adsorbents have been shownuseful in the removal of antibiotics and other antimicrobials from bloodsamples. The removal of these inhibitors in medical samples allows forrecovery and faster detection of microorganisms so that microbialidentification and accurate antibiotic susceptibility testing can beperformed.

Melnick et al., U.S. Pat. No. 4,145,304, herein incorporated byreference, describes the use of synthetic anionic exchange and nonionicresins to remove antimicrobials, including antibiotics, from bodyfluids, thus allowing for recovery of pathogens using standard culturetechniques. The resins described are coated with a nonionic detergent inorder to selectively remove charged antibiotics while inhibitingadherence of bacteria to the resins. After treatment of the sample withthe resin, the eluate is cultured in a growth media. The degree ofbinding of antibiotics by the resins is indicated to be dependent on thetotal exchange capacity, pore diameter, and surface area of the resin.

Waters, U.S. Pat. No. 4,632,902, herein incorporated by reference,describes an improvement over Melnick by incorporating ion exchangeresins and non-functional adsorbent resins directly into the growthmedium. Inhibitors removed include antibiotics administered to patientsand naturally occurring inhibitors contained in serum, plasma, and lysederythrocytes. The resins are not coated with a nonionic detergent orsurfactant before use and the pore size of the resin is not critical.

Thorpe et al., U.S. Pat. Nos. 5,162,229 and 5,314,229, hereinincorporated by reference, describe the use of resin and non-resinousadsorbents to neutralize, bind, or inhibit antimicrobial substances thatmay be present in a biological sample. The resin and non-resinousadsorbents described include, aluminum oxide, colloidal native hydratedaluminum silicates, crystalline hydrated alkali-aluminum silicates,silica, siliceous frustules, fragments of various species of diatoms,amorphous carbon, ion exchange resins, non-functional polymeric resinadsorbents, polystyrene resin cross-linked with divinyl benzene andcombinations thereof.

Although synthetic resins and non-resinous adsorbents are known toremove inhibitory substances in cultures containing body fluids, theseresins and non-resinous adsorbents may be ineffective in neutralizing orremoving some types of antibiotics. For example, some β-lactams arecommonly used in the treatment of sepsis and their presence in bloodsamples can interfere with the recovery, detection and identification ofthe microorganism responsible for the sepsis. In particular, resins andnon-resinous adsorbents currently used in the art to removeantimicrobials from blood samples have proven largely ineffective inneutralizing carbapenems. It is therefore desirable to find other meansfor the neutralization or inhibition of antibiotics in body fluids andnon-body fluid samples, such as foods and industrial products.

The present invention provides a means for the neutralization orinhibition of antimicrobials in test samples, while helping to retainthe components of the medium necessary to recover and detectmicroorganisms in a rapid manner. By finding a means for theneutralization and/or inactivation of β-lactams (e.g., carbapenems) thatpreviously could not be effectively neutralized or inactivated in aculture medium, the present invention solves a long-felt need in theart.

SUMMARY OF THE INVENTION

In one aspect, the present invention involves a means or methodinvolving the use of one or more primary amine-containing compounds toneutralize, bind, inhibit or otherwise inactivate an antimicrobialsubstance (e.g., one or more antibiotics) in a growth or culture medium.In one embodiment, the present invention is directed to theneutralization, inhibition or inactivation of one or more antibioticswith one or more primary amine-containing compounds in culture medium(e.g., a blood culture).

The present invention is also directed to a method for theneutralization and/or inactivation of an antimicrobial in a culturemedium (e.g., a blood culture) comprising adding one or more primaryamine-containing compounds to a culture medium which is capable ofsupporting growth of microorganisms, wherein said one or more primaryamine-containing compounds are present in an amount effectiveneutralizes and/or inactivates any carbapenems present in said culturemedium. In one embodiment, the antimicrobial is a carbapenem. In anotherembodiment, the primary amine is a non-thiol containing primary amine.

In another aspect, the present invention is directed to a method forenhanced recovery and detection of microorganisms in culture, the methodcomprising: (a) preparing culture medium; (b) adding to the medium atleast one primary amine-containing compound in amounts that areeffective for neutralizing, binding or inhibiting antimicrobialsubstances in the culture medium; (c) inoculating the medium with asample; and (d) incubating and determining the results. In oneembodiment, the antimicrobial is a carbapenem. In another embodiment,the primary amine is a non-thiol containing primary amine.

In still another aspect, the present invention is directed to a methodfor the diagnosis of an infection caused by a microorganism, comprisingthe steps of: (a) obtaining a specimen sample or test sample for whichthe presence or absence of a microorganism is to be determined, andwherein said specimen or test sample may contain one or moreantimicrobials that may interfere with the growth and/or detection ofthe microorganism; (b) adding said specimen sample or test sample to aculture medium, said culture medium comprising at least one primaryamine-containing compound in an amount that is effective forneutralizing, binding or inhibiting said one or more antimicrobials; and(c) analyzing said culture for the presence of said microorganism,wherein detection of the presence of said microorganism indicates apositive diagnosis for said infection. In one embodiment, theantimicrobial is a carbapenem. In another embodiment, the primary amineis a non-thiol containing primary amine. In still another embodiment,detection of said microorganism comprises detection of the growth ofsaid microorganism in said culture medium.

In yet another aspect, the present invention is directed to a device fordetecting microorganisms suspected of being in a specimen comprising asealable specimen container comprising an internal chamber in which thespecimen may be cultured in a culture medium, said culture mediumcontaining at least one primary amine-containing compound, wherein saidprimary amine-containing compound neutralizes and/or inactivatesantibiotics that may be present in said specimen.

In still another aspect, the present invention is directed to a kit fordetecting microorganisms in a test sample, the kit comprising: (1) asealable specimen container, having an internal chamber comprising aculture medium and in which a test sample, for which the presence orabsence of a microorganism is to be determined, may be cultured; and (2)a supplement comprising one or more primary amine-containing compounds.In accordance with this aspect of the present invention, the supplementcan be added to the culture medium contained in the sealable specimencontainer, thereby providing the one or more primary amine-containingcompounds in an amount that is effective for neutralizing, binding orinhibiting any antimicrobials that may be present in said culturemedium. A test sample can be added to the culture medium, concurrentlywith, or after addition of the supplement to the culture medium, thesupplement providing the one or more primary amine-containing compoundsin an amount that is effective for neutralizing, binding or inhibitingany antimicrobials that may be present in said test sample.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A—is a Size Exclusion Chromatography (SEC) Chromatogram ofimipenem in the presence of cysteine after a reaction time of 0 min.

FIG. 1B—is a Size Exclusion Chromatography (SEC) Chromatogram ofimipenem in the presence of cysteine after a reaction time of 20 min.

FIG. 1C—is a Size Exclusion Chromatography (SEC) Chromatogram ofimipenem in the presence of cysteine after a reaction time of 60 min.

FIG. 1D—is a Size Exclusion Chromatography (SEC) Chromatogram ofimipenem in the presence of cysteine after a reaction time of 90 min.

FIG. 2A—is a Size Exclusion Chromatography (SEC) Chromatogram ofimipenem, without cysteine after a reaction time of 0 min.

FIG. 2B—is a Size Exclusion Chromatography (SEC) Chromatogram ofimipenem, without cysteine after a reaction time of 20 min.

FIG. 2C—is a Size Exclusion Chromatography (SEC) Chromatogram ofimipenem, without cysteine after a reaction time of 60 min.

FIG. 2D—is a Size Exclusion Chromatography (SEC) Chromatogram ofimipenem, without cysteine after a reaction time of 90 min.

FIG. 3—is a boxplot showing the effect of cysteine on the recovery of S.aureus in the presence of imipenem.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for detecting the presence ofmicroorganisms in a specimen or test sample containing or suspected ofcontaining microorganisms. In accordance with this invention, themethods involve a chemical neutralization method involving the use ofprimary amine-containing compounds to neutralize, bind, inhibit orotherwise inactivate an antimicrobial substance (e.g., antibiotics) in agrowth or culture medium. In one embodiment, the present invention isdirected to the neutralization, inhibition or inactivation of one ormore antibiotics with a primary amine-containing compound in bloodculture medium. In some embodiments, the primary amine-containingcompound may be a non-thiol containing compound and/or a hydroxylamine.

Samples that may be tested by the methods of the invention include bothclinical and non-clinical samples in which microorganism presence and/orgrowth is or may be suspected, as well as samples of materials that areroutinely or occasionally tested for the presence of microorganisms.Test samples can typically range from about 0.5 ml to about 50 ml, fromabout 1 ml to about 10 ml, or from about 2 ml to about 5 ml.

Clinical specimens or specimen samples that may be tested include anytype of sample typically tested in clinical or research laboratories,including, but not limited to, blood, serum, plasma, blood fractions,joint fluid, urine, semen, saliva, feces, cerebrospinal fluid, gastriccontents, vaginal secretions, tissue homogenates, bone marrow aspirates,bone homogenates, sputum, aspirates, swabs and swab rinsates, other bodyfluids, and the like. In one embodiment of the present invention,samples are obtained from a subject (e.g., a patient) having orsuspected of having a microbial infection. In one embodiment, thesubject has or is suspected of having septicemia, e.g., bacteremia orfungemia. The sample may be a blood sample taken directly from thesubject.

Non-clinical samples that may be tested also include substances,encompassing, but not limited to, foodstuffs, beverages,pharmaceuticals, cosmetics, water (e.g., drinking water, non-potablewater, and waste water), seawater ballasts, air, soil, sewage, plantmaterial (e.g., seeds, leaves, stems, roots, flowers, fruit), bloodproducts (e.g., platelets, serum, plasma, white blood cell fractions,etc.), donor organ or tissue samples, biowarfare samples, and the like.The method is also particularly well suited for real-time testing tomonitor contamination levels, process control, quality control, and thelike in industrial, commercial, and/or clinical settings.

A first aspect of the invention relates to methods for theneutralization and/or inactivation of antibiotics using one or moreprimary amine-containing compounds. The method comprises adding one ormore primary amine-containing compounds to a growth or culture medium inan effective amount to neutralize, bind, and/or inhibit one or moreantibiotics present, or suspected of being present, in a test sample.The one or more primary amine-containing compounds can be added to thegrowth or culture media prior to, or concurrently with, innoculation ofthe growth or culture media with a test sample. Although, not wishing tobe bound by theory, it is believed that the use of a primaryamine-containing compound provides a chemical means for neutralizationof antimicrobials, whereby the primary amine interacts or binds with theβ-lactam ring structure of the antibiotic. The primary amine may carryout a nucleophilic attack on the β-lactam ring forming a covalentcomplex leading to the inactivation and/or neutralization of theβ-lactam antibiotic. After inoculation, the growth or culture media andtest sample can be incubated for a sufficient time and at a sufficienttemperature to allow for the growth and detection of any microorganismthat may be present in the test sample. Growth can be detected by anyknown means in the art. For example, growth can be detected using aBacT/ALERT® or BacT/ALERT® 3D systems (bioMerieux, Inc.). The time andtemperature required for growth of the microorganism are largely speciesspecific, but typically will be from about 1 hour to about 48 hours andfrom about 30° C. to about 42° C.

An “effective amount” means the use of a sufficient amount of a compoundto neutralize, bind, and/or inhibit the activity of one or moreantibiotics present, or suspected of being present, in a test sample orculture media. An “effective amount” can be an amount sufficient toproduce a measurable inhibition of one or more antibiotics present in atest sample or medium. Inhibition of antibiotics can be measured invitro by high-performance liquid chromatography (HPLC), or by othermethods known to one skilled in the art. An “effective amount” can alsobe an amount sufficient to show detectable microorganism growth in atest sample or culture media containing an antibiotic whose activitywould otherwise suppress or eliminate detectable growth. An “effectiveamount” may not need to be an amount that would totally eliminate theactivity of one or more antibiotics present, or suspected of beingpresent in the growth or culture media. Rather, in the practice of thepresent invention, the use of an “effective amount” of one or moreprimary amine-containing compounds in a growth or culture media allowsfor the growth or cultivation of microorganisms that would otherwise besurpressed or eliminated from the presence of one or more antibioticsthat are neutralized, bound, and/or inhibited by the primaryamine-containing compound, in accordance with the present invention.Typically, an “effective amount” of one or more primary amine-containingcompounds is an amount allowing for a final concentration in the growthof culture media of from about 0.1 g/L to about 20 g/L, from about 0.5g/L to about 10 g/L, or from about 1 g/L to about 5 g/L.

In one embodiment, the present invention is a method for enhancedrecovery and detection of microorganisms in culture, the methodcomprising: (a) preparing culture medium; (b) adding to the medium atleast one primary amine-containing compound in amounts that areeffective for neutralizing, binding or inhibiting antimicrobialsubstances in the culture medium; (c) inoculating the medium with asample to be tested; and (d) incubating and determining the results. Inanother embodiment, the present invention is also directed to a methodfor the neutralization and/or inactivation of a carbapenem in a bloodculture comprising adding a primary amine-containing compound to a bloodculture medium which is capable of supporting growth of microorganisms,wherein said primary amine-containing compound neutralizes and/orinactivates any carbapenems present in said culture medium.

In another aspect, the present invention relates to a method for thediagnosis of an infection caused by a microorganism, comprising thesteps of: (a) obtaining a test sample for which the presence or absenceof a microorganism is to be determined, and wherein said test sample maycontain one or more antimicrobials which may interfere with the growthand/or detection of the microorganism; (b) adding said test sample to aculture medium, said culture medium comprising at least one primaryamine-containing compound in an amount that is effective forneutralizing, binding or inhibiting said one or more antibiotics; and(c) analyzing said culture for the presence of said microorganism (e.g.,detection of said microorganism or the growth of said microorganism),wherein a finding of the presence of said microorganism indicates apositive diagnosis for said infection. After adding the test sample tothe growth or culture media, the growth or culture media and test samplecan be incubated for a sufficient time and at a sufficient temperature,as is well known to those skilled in the art, to allow for the growthand detection of any microorganism that may be present in the testsample. Growth can be detected by any known means in the art. Forexample, growth can be detected using a BacT/ALERT® or BacT/ALERT® 3Dsystems (bioMerieux, Inc.). Growth readings can be taken continuously ora give time intervals.

In general, any known antibiotic can be neutralized using the methods ofthe present invention. In one aspect, the present invention is directedto the use of a primary amine-containing compounds to neutralize and/orinactivate a β-lactam antibiotic. β-lactam antibiotics are a broad classof antibiotics that include any antibiotic agent that contains aβ-lactam nucleus in its molecular structure. β-lactam antibioticsinclude, but are not limited to, penicillins, penicillin derivatives,cephalosporins, monobactams, carbapenems, and β-lactamase inhibitors.

In one embodiment, primary amine-containing compounds can be used toneutralize and/or inactivate penicillin and penicillin derivativeantibiotics. Penicillins are a class of β-lactam antibiotics known aspenams. Penams are a group of antibiotics sharing a similar coreskeleton (R—C₉H₁₁N₂O₄S, where R is a variable side chain). Penicillinsand penicillin derivatives include, for example, benzathine penicillin,benzylpenicillin (penicillin G), phenoxymethylpenicillin (penicillin V),procaine penicillin, oxacillin, methicillin, nafcillin, cloxacillin,dicloxacillin, flucloxacillin, temocillin, amoxycillin, ampicillin,azlocillin, carbenixillin, mezlocillin, and piperacillin.

In another embodiment, primary amine-containing compounds can be used toneutralize and/or inactivate cephalosporins and cephalosporinderivatives. Cephalosporins are a group of β-lactam antibiotics sharinga core skeleton comprising 7-aminocephalosporanic acid. Cephalosporinsand cephalosporin derivatives include, for example, cephalexin,cephalothin, cefazolin, cefaclor, cefuroxime, cefamandole, cefotetan,cefoxitin, ceftrizxone, cefotaxime, cefpodoxime, ceftazidime, cefepime,and cefpirome.

In yet another embodiment, primary amine-containing compounds can beused to neutralize and/or inactivate carbapenem and carbapenemderivatives. Carbapenems are a class of β-lactam antibiotics with abroad spectrum of antibacterial activity, and have a structure thatrenders them resistant to β-lactamases. Carbapenems and carbapenemderivatives include imipenem, meropenem, ertapenem, doripenem,panipenem, betamipron, biapenem, and PZ-601. The carbapenems aresomewhat structurally similar to the penicillins, but the sulfur atom inposition 1 of the structure has been replaced with a carbon atom, andhence the name of the group, the carbapenems. Nevertheless, thisseemingly subtle structural difference can lead to dramatic differenteffects in chemical and biological activity.

In still another embodiment, primary amine-containing compounds can beused to neutralize and/or inactivate β-lactamase inhibitors andderivatives of β-lactamase inhibitors. β-lactamase inhibitors andderivatives include, for example, calvulanic acid, tazobactam andsulbactam.

As previously mentioned, in accordance with this invention, the primaryamine-containing compound can be added to a growth or culture media. Theprimary amine-containing compound can be added directly to the mediaprior to, or concurrently, with inoculation of the medium with a sampleto be tested. In another embodiment, the primary amine-containingcompound can be added directly to the sample to be tested, prior to thesample being added to the growth or culture medium.

The use of a growth or culture media (or medium) for the cultivation ofmicroorganisms is well known. A suitable growth or culture mediumprovides the proper nutritional and environmental conditions for growthof microorganisms and should contain all the nutrients required by themicroorganisms which are to be cultivated. For example, a typicalmicrobiological culture medium should contain water, a carbon source, anitrogen source, vitamins, trace elements such as potassium, magnesium,calcium and iron, and minerals, such as sulfur and phosphorous.Typically, these needs are supplied from a number of sources. Otherfactors for suitable propagating conditions may include pH, temperature,aeration, salt concentration and osmotic pressure of the medium.

In addition, it is known that certain growth factors may be required. Agrowth factor is an organic compound which a microorganism must containin order to grow but which it is typically unable to synthesize. Manymicroorganisms, when provided with the nutrients listed above, are ableto synthesize all of the organic constituents of their protoplasm,including amino acids, vitamins, purines and pyrimadines, fatty acidsand other compounds. Typically, each of these essential compounds can besynthesized by a discrete sequence of enzymatic reactions, where eachenzyme is produced under the control of a specific gene. However, for avariety of reasons some microorganism cannot synthesis one or more ofthese growth factors and must then obtain that compound from theenvironment. Required growth factors may include, but are not limitedto, amino acids, vitamins, purines and pyrimadines, fatty acids andother required compounds for growth.

The growth or culture media used in the practice of the presentinvention can be any known growth of culture media for the cultivationof microorganisms. Typically, the culture medium of the presentinvention comprises a liquid nutrient medium or nutrient broth. Theculture medium or nutrient broth of the present invention typicallycomprises one or more known nutrients, for example, the culture mediummay contain one or more carbon sources (e.g., glycerol), nitrogensources (e.g., ammonia salts), sugars, salts (e.g., K⁺, Mg²⁺, Ca²⁺,Zn²⁺), nutrients, and/or water. In one embodiment, the culture medium ofthe present invention may further comprise one or more of potassiumsalts, sodium salts, sodium glutamate, sodium citrate, ammonium sulfate,pyridoxine, ferric ammonium citrate, magnesium sulfate, zinc sulfate,copper sulfate, biotin, calcium chloride, or combinations thereof. Inanother embodiment, general purpose medias can be used, include, forexample, tryptic soy broth, brain heart infusion broth, Columbia broth,and Brucella broth.

In general, any known primary amine-containing compound can be used inthe practice of the present invention. As discussed hereinabove, one ormore primary amine-containing compounds can be added to the growth orculture media prior to, or concurrently with, inoculation of the growthor culture media with a test sample. In one embodiment, the primaryamine-containing compound can be added as a supplement to the culturemedium prior to the inoculation of the primary amine-containing culturemedium with a sample to be tested. In an alternative embodiment, theprimary amine-containing compound can be added directly to the testsample prior to inoculation of the culture medium with the primaryamine-containing test sample. Useful primary amines include, but are notlimited to, a primary amine of the formula R—NH₂ where R is a linear orbranched alkyl, aryl, alkaryl, or aralkyl group having between about 1and about 20 carbon atoms. Preferred R groups include, withoutlimitation, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl,isobutyl, hexyls (linear or branched), hepyls, octyls, nonyls, decyls,phenyl, benzyl, methyl substituted phenyls, or mixtures or combinationsthereof. In one embodiment, the primary amine is a hydroxylamineExemplary primary amines may include methylamine, ethanolamine,trisamine, propylamine, 2-aminoheptane, 2-amino-2-methyl-1,3propanediol, 2-amino-2-methyl-1-propanol, n-amylamine, benzylamine,1,4-butanediamine, n-butylamine, cyclohexylamine, ethylamine,ethylenediamine, α-methylbenzylamine, phenethylamine, isopropylamine,butylamine, sec-butylamine, iso-butylamine, andtris(hydroxymethyl)aminomethane.

In some embodiments, organic primary amines may be preferred. Suitableorganic primary amines may include aliphatic, cycloaliphatic,aliphatic/aromatic, aromatic amines, diamines and/or polyamines, such asmethylamine, ethylamine, butylamine, stearylamine, aniline,halogen-substituted phenylamines (e.g., 4-chlorophenylamine),1,4-diaminobutane, 1,6-diaminohexane, 1,8-diamino-hexane,1-amino-3,3,5-trimethyl-5-aminocyclohexane, lysine ethyl ester, lysineaminoethyl ester, 1,6,11-triaminoundecane or 1,5-naphthylenediamine,1,4-diaminobenzene, p-xylylenediamine, perhydrogenated 2,4- and/or2,6-diaminotoluene, 2,2′-, 2,4′- and/or 4,4′-diaminodicyclohexylmethane,2,4-, 2,6-diaminotoluene and their mixtures, 4,4′-, 2,4′- and/or2,2′-diphenylmethanediamine and their mixtures, as well as highermolecular weight isomeric, oligomeric or polymeric derivatives of theseamines and polyamines. Other possible amines are known from the priorart. Preferred amines for the present invention are the diamines andpolyamines of the diphenylmethane series (MDA, monomeric, oligomeric andpolymeric amines), 2,4-, 2,6-diaminotoluene (TDA, toluoylenediamines),for example technical mixtures of 2,4-, 2,6-diaminotoluene (TDA,toluoylenediamines) in a weight ratio of 80:20, isophorone diamine andhexamethylenediamine. The corresponding isocyanatesdiisocyanatodiphenylmethane (MDI, monomeric, oligomeric and polymericisocyanates), toluene diisocyanate (TDI), hexamethylene diisocyanate(HDI) and isophorone diisocyanate (IPDI) are obtained in thephosgenation.

In other embodiments, the primary amine can be an amino acid. Exemplaryamino acids include, but are not limited to, alanine, arginine,asparagine, aspartate, cysteine, glutamate, glutamine, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine, and valine.

In some embodiment, the use of a hydroxylamine may be preferred. As oneof skill in the art is well aware, hydroxylamines generally includecompounds having the formula NH₂OH, and salts thereof. Exemplifieduseful hydroxylamines include, but are not limited to, methanolamine,ethanolamine, propanolamine, butanolamine, pentanolamine, hexanolamine,heptanolamine, octanolamine, nonanolamine and decanolamine.

Thiol groups or sulfhydryl groups tend to be scavengers of oxygen, andthus, may interfere with components of a growth or culture media and/ormicroorganism growth in a growth or culture media. As such, in someembodiments the use of one or more non-thiol containing primary aminesmay be preferred. As one of skill in the art is well aware, non-thiolcontaining primary amines may include any known primary amine-containingcompounds that does not contain a thiol group. Exemplary non-thiolcontaining primary amines include, but are not limited to, methylamine,ethanolamine, trisamine, propylamine, 2-aminoheptane,2-amino-2-methyl-1,3 propanediol, 2-amino-2-methyl-1-propanol,n-amylamine, benzylamine, 1,4-butanediamine, n-butylamine,cyclohexylamine, ethylamine, ethylenediamine, α-methylbenzylamine,phenethylamine, isopropylamine, butylamine, sec-butylamine,iso-butylamine, and tris(hydroxymethyl)aminomethane. In yet otherembodiment, the use of a non-thiol containing amino acid may bepreferred. Useful non-thiol containing amino acids include, but are notlimited to, alanine, arginine, asparagine, aspartate, glutamate,glutamine, glycine, histidine, isoleucine, leucine, lysine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, andvaline.

In another embodiment, the primary amine can be bound to, or immobilizedon, a solid support or a polymeric carrier, and the primaryamine-support complex added to a culture medium or bottle, as discussedherein. In general, the support may be any material on which a bindingpartner can be immobilized, such as nitrocellulose, silica, polystyrene,polypropylene, polyvinyl chloride, EVA, glass, carbon, glassy carbon,carbon black, carbon nanotubes or fibrils, platinum, palladium, gold,silver, silver chloride, iridium, or rhodium. In one embodiment, thesolid support can be, polymeric carriers, for example modified silicagel, glass, especially “controlled pore glass”, polyester, polyamide,polyvinyl alcohol, polysiloxane, polystyrene or the like. In anotherembodiment, useful solid supports may include, but are not limited to,silica and neutral macroporous resins such co-polymers of styrene anddivinylbenzene.

In another aspect of the present invention, the growth or culture mediamay further comprise one or more adsorbents for the neutralization,inhibition and/or removal of additional antimicrobial substances thatmay be present in a test sample. Generally, antimicrobial substancesinclude, among others, antibiotics, antibiotics in body fluid samples,preservatives, bacteriostats, bactericides, and any toxic by-productsproduced during the preparation of culture media. Antimicrobialsubstances also include naturally occurring components in blood such ascomplement and antibodies.

The term “adsorbent” for the purposes of this application, includes alladsorbent materials that neutralize, bind, and inhibit antimicrobialsubstances. These adsorbents include resins and non-resinous adsorbentsas defined in U.S. Pat. Nos. 4,145,304; 4,632,902; 5,162,229; and5,314,229.

As used herein, “resin” is a subclass of adsorbents, and is furtherdefined to include naturally occurring and synthetic resins, for exampleion exchange resins, non-functional polymeric resin adsorbents and, inparticular, polystyrene resins cross-linked with divinyl benzene. Usefulresins can include, but are not limited to, those disclosed in U.S. Pat.Nos. 4,145,304 and 4,632,902. For example, useful resins may includesodium, hydrogen and ammonium charged cation exchange resins such as:BIO REX AG 50W—X₂, X₄, X₆, X₈, X₁₀, X₁₂, and X₁₆ from BIO-RADLaboratories, DOWEX 50W—X₂, X₄, X₆, X₈, X₁₀, X₁₂, and X₁₆ from DowChemical Company and Rexyn 101 from Fisher Scientific Co., all of whichare strong acid polystyrene resins having SO³⁻ functional group. Usefulnon-functional resins may include XAD resins manufactured by Rohm & Haasand SM resins sold by BioRad. For example, chloride, formate, acetateand hydroxide, charged anion exchange resins have generally been foundto be suitable. Specifically, chloride charged anionic exchange resinsin combination with adsorbent resins sold under the following trademarksmay be effective in the practice of the invention. DOWEX 1-X8 from DowChemical Company, DUOLITE A-109 from Diamond Shamrock Company andAMBERLITE IRA400 from Rohm & Haas, all of which are strong base resinshaving polystyrene quaternary ammonium functional groups; DUOLITE A-7from Diamond Shamrock Company and AMBERLITE IR45 from Rohm & Haas, whichare weakly basic and have tertiary amine functional groups. In someembodiments, the cation and anion exchange resins may preferably be usedin combination with a non-functional resin such as the XAD resins fromRohm & Haas and SM resin from BioRad, particularly XAD-4 resin which isa nonfunctional copolymer of styrene and divinyl benzene.

As used herein “non-resinous adsorbents” are another subclass ofadsorbents and are defined as naturally occurring and syntheticnon-resin adsorbents and molecular sieves that can be used forclarifying, deodorizing, decolorizing, and filtering. Some of thesenon-resinous adsorbents are the same as those used during the productionof antibiotics to remove antibiotics from culture medium growingantibiotic-producing bacteria. Useful non-resinous adsorbents includethose disclosed in U.S. Pat. Nos. 5,162,229 and 5,314,229. For example,useful non-resinous adsorbents include various forms of 1) aluminumoxide (alumina), 2) colloidal native hydrated aluminum silicates(clays), such as bentonite, kaolin, and fuller's earth, 3) crystallinehydrated alkali-aluminum silicates (sodium or calcium zeolites), 4)silica (silica gel, silica beads) such as Davisil, 5) siliceousfrustules and fragments of various species of diatoms (infusorial earth,diatomaceous earth) such as Celite™ (Manville Products Corporation,Denver, Colo., USA) and 6) amorphous carbon (in particular, activatedcarbon) such as Carboraffin, Norit™ (American Norit Company Inc.,Jacksonville, Fla., USA), Opocerbyl, and Ultracarbon. Naturallyoccurring adsorbent activated charcoal, which has been used to preventthe lethal effects of oxidation in transport media and growth media, canalso be used. This media has been used for the transport of fastidiousorganisms such as Neisseria gonorrhoeae and the cultivation ofLegionella species. Non-resinous adsorbents do not require pre-treatmentwith a surfactant in order to function. Treatment with surfactants mayeven decrease the adsorptive capabilities of these materials.

The use of adsorbents at an appropriate ratio to medium may also removetoxic by-products produced in autoclaved media and still provide anoptimal nutritious culture medium while maintaining the ability toneutralize antimicrobial substances. The resins and/or non-resinousadsorbents may be present in the culture media from about 0.1 g to about10 g per bottle.

The present adsorbents are not limited to use in a device or culturebottle. They may be added to any standard culture media, which is theninoculated with a sample, incubated at the correct temperature for anappropriate time for the type of sample being tested, while usuallyshaken or rocked in order to expose more surface area of the adsorbentto the liquid, to better contact any organisms present with nutrientsand to avoid areas of high concentration of metabolic by-products. Thetemperatures and time periods needed for the determination ofmicroorganism growth are well known to those skilled in the art and varysomewhat among different types of organisms.

In another aspect, the present invention is directed to a device fordetecting microorganisms suspected of being in a test sample or specimencomprising a sealable container comprising an internal chamber in whichthe test sample or specimen may be cultured in a growth or culturemedium, said growth or culture medium containing at least one primaryamine-containing compound, wherein said primary amine-containingcompound neutralizes, binds and/or inactivates one or more antibiotics(e.g., carbapenems) present in said specimen. In one embodiment, thedevice is a blood culture bottle, as described, for example, in U.S.Pat. Nos. 5,094,955 and 5,162,229. In accordance with this aspect of thepresent invention, a test sample is introduced into the device, and thedevice is incubated until either positive growth is detected or,generally, until 5-7 days have passed and no growth is detected.

In another embodiment, the primary amine-containing compound can beincluded in a supplement that can be added to the culture medium priorto, or concurrently with inoculation of the culture medium with thesample to be tested. In an alternative embodiment, the primaryamine-containing supplement can be added directly to the test sample,prior to inoculation of the bottle and culture media with the primaryamine-containing test sample. The supplement may further comprise one ormore nutrients and/or components known to those of skill in the art asbeing beneficial to the cultivation of microorganisms. For example, thesupplement of the present invention may additionally comprise of one ormore sugars, carbon sources, nitrogen sources, minerals, salts, aminoacids, vitamins, purines and pyrimadines, fatty acids and othercompounds. After addition of the supplement and inoculation of theculture medium with the sample to be tested, the culture media andsample can be cultivated for a sufficient time and at a sufficienttemperature to allow for the growth and detection of any microorganismthat may be present in the test sample.

In still another aspect, the present invention is directed to a kit fordetecting microorganisms in a test sample, the kit comprising: (1) asealable specimen container, having an internal chamber comprising aculture medium and in which a test sample, for which the presence orabsence of a microorganism is to be determined, may be cultured; and (2)a supplement comprising one or more primary amine-containing compounds.In accordance with this aspect of the present invention, the supplementcan be added to the culture medium contained in the sealable specimencontainer, thereby providing the one or more primary amine-containingcompounds in an amount that is effective for neutralizing, binding orinhibiting any antimicrobials that may be present in said culturemedium. A test sample can be added to the culture medium, concurrentlywith, or after addition of the supplement to the culture medium, thesupplement providing the one or more primary amine-containing compoundsin an amount that is effective for neutralizing, binding or inhibitingany antimicrobials that may be present in said test sample. Thesupplement of the kit further comprises a second container or vialcomprising the one or more primary amine-containing compounds. In oneembodiment, the second container or vial comprises the one or moreprimary amine-containing compounds suspended in a stabilization buffer.Stabilization buffers are well known to those skilled in the art. Inanother embodiment, the one or more primary amine-containing compoundspresent in said container or vial can be lyophilized. In accordance withthis embodiment, the lyophilized one or more primary amine-containingcompounds may be re-suspended in a re-suspension buffer or stabilizationbuffer prior to being added to the culture medium in the specimencontainer. In yet another embodiment, the kit of the present inventionmay further provide a third container or vial comprising saidre-suspension or stabilization buffer.

As those of skill in the art are aware, the presence of microorganismscan be determined by detecting or measuring changes in the pH of thespecimen or the production of CO₂ within a specimen using a disposablesensor affixed to the interior surface of the container, as described,for example, in U.S. Pat. Nos. 4,945,060 and 5,164,796, which areincorporated herein by reference. According to the '060 and '796disclosures, microorganisms can be detected in the presence ofinterfering materials, such as large concentrations of red blood cells,through non-radiometric and non-invasive means. As the level of pHand/or CO₂ within the specimen changes, the light reflecting and/orabsorbing characteristics of the disposable sensor will altercorrespondingly. The quantity of alteration of the reflective propertiesof the sensor is detected by an emission and receiving mechanism whichsupplies signals to a device for monitoring the quantity of visiblereflection/absorption and the rate of change. The rate and quantity isthen analyzed to predict and determine the presence of microbial growthwithin the specimen or sample. The sensor can be sampled and/ormonitored continuously or at frequent time intervals allowing for thecollection of a detailed characteristic of the quantity and rate ofsensor change. As described in the art, the sensor means may comprise amembrane and an indicator medium, the indicator medium being selectedfor its ability to exhibit a detectable change when exposed to productsof an organism's metabolic activity. As known by those skilled in theart, the changes in the appearance of the sensor means can becontinuously monitored from the exterior of the container through atransparent section of the container.

This device or blood culture bottle may also include materials in theculture medium such as the resinous materials, as described in U.S. Pat.Nos. 4,145,304 and 4,632,902, and non-resinous adsorbent materials, asdescribed in U.S. Pat. Nos. 5,162,229 and 5,314,229, or combinationsthereof, that neutralize, bind, or inhibit any other antimicrobialsubstances that may be present in the test sample of culture media. Theresins and/or non-resinous adsorbents may be present in the device fromabout 0.1 g to about 10 g per bottle.

The present invention can be embodied in many different forms and shouldnot be construed as limited to the embodiments set forth herein. Rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey the scope of the invention to thoseskilled in the art. For example, features illustrated with respect toone embodiment can be incorporated into other embodiments, and featuresillustrated with respect to a particular embodiment can be deleted fromthat embodiment. In addition, numerous variations and additions to theembodiments suggested herein will be apparent to those skilled in theart in light of the instant disclosure, which do not depart from theinstant invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

The following examples are given to further illustrate features of theinvention, but are not intended to limit the scope of the invention inany way.

EXAMPLES Example 1 Use of Cysteine to Neutralize Imipenem

A stock solution of USP grade imipenem was prepared by dissolving 1.8grams in 10 mL of 14 mM K₂HPO₄ (pH 7.0) in a glass screw cap tube. Astock solution of cysteine was prepared by dissolving 31.3 mg in 1.08 mLof 14 mM K₂HPO₄ in a 2 mL polypropylene centrifuge tube.

An imipenem reaction without cysteine was prepared by combining 2.2 mLof the stock imipenem solution with 7.8 mL of 14 mM K₂HPO₄ to a finalconcentration of 40 μg/mL. The total 10 mL volume was then added to aBacT/Alert culture bottle containing resin adsorbents. The bottle wasbriefly agitated to mix and 500 μL of volume was removed for analysis.The bottle was then placed in a BacT/Alert® 3D instrument (bioMerieux,Inc., Missouri, USA) at 36° C. and additional 500 μL samples wereremoved at 20, 40, 60 and 90-minute intervals for analysis (see FIGS.2A-2D).

An imipenem reaction with cysteine was prepared by combining 2.2 mL ofthe stock imipenem solution with 7.8 mL of 14 mM K₂HPO₄ to a finalconcentration of 40 μg/mL. The total 10 mL volume was then added to aBacT/Alert culture bottle containing resin adsorbents. A 500-uL sampleof the cysteine stock solution was then added to a final cysteineconcentration of 4 mM. The bottle was briefly agitated to mix and 500 μLof volume was removed for analysis. The bottle was then placed in aBacT/Alert® 3D instrument (bioMerieux, Inc., Missouri, USA) at 36° C.and additional 500 μL samples were removed at 20, 40, 60 and 90-minuteintervals for analysis (see FIGS. 1A-1B).

Reaction samples were analyzed on an HPLC equipped with aBioSep™-SEC-2000 (Phenomenex) size exclusion HPLC column with a mobilephase of 100 mM Na₂HPO₄, ph 6.5 at a 1-mL/minute flow rate. Reactionswere monitored at 300 nm and the total analysis time per sample was 16minutes.

The bottle containing both imipenem and cysteine showed a reduction ofimipenem of approximately 70%, 85%, 89% and 91% after 20, 40, 60, and 90minutes, respectively (see Table 1). However, after 90 minutes thebottle containing only imipenem showed no reduction in imipenem (seeFigure Table 2).

TABLE 1 Imipenem + Cysteine Reaction Reaction time Peak area at % ChangeFrom (min) 10.88 min T = 0 0 1619342 — 20 481363 −70 40 251741 −85 60179565 −89 90 146769 −91

TABLE 2 Imipenem + No Cysteine Control Reaction time Peak area at %Change From (min) 10.88 min T = 0 0 1619342 — 20 1756343 +8.5 40 1795587+11 60 1798536 +11 90 1737014 +7

Example 2 Growth Performance in Resin Bottle Containing Imipenem withand without Cysteine

An imipenem solution was prepared by dissolving 4.0 mg of imipenem into100 mL of phosphate buffered saline (PBS), pH 7.0, and sterile filteredthrough a 0.2μ filter. Cysteine was prepared by dissolving 300 mg into10 mL PBS and sterile filtered through a 0.2μ filter to achieve a finalconcentration in BacT/Alert® bottles (bioMerieux Inc, Missouri, USA) of4 mM with the addition of 500 μL. A culture of S. aureus was preparedafter overnight incubation to a final concentration of 30-300 CFU(colony forming units)/500 μL.

BacT/Alert® culture bottles (bioMerieux Inc, Missouri, USA) containingresin adsorbents were prepared for evaluation in triplicate as follows:

Growth controls were prepared by adding 10 mL of sterile filtered PBSalone or 10 mL of sterile filtered PBS plus 500 μL of cysteine solution.A control for the imipenem activity was prepared by adding 10 mL ofimipenem solution. The imipenem/cysteine reaction was prepared by adding10 mL of imipenem solution plus 500 μL of cysteine solution. Each bottlereceived 500 μL of the S. aureus culture and was then placed in aBacT/Alert 3D® instrument (bioMerieux Inc, Missouri, USA) at 36° C. andmonitored for growth over five days.

After 5 days all the bottles containing imipenem without cysteine werenegative for S. aureus growth. However, within 28 hours 2 of the 3bottles containing both imipenem and cysteine were positive for S.aureus growth.

1. A method for the neutralization and/or inactivation of a carbapenemin a culture medium, the method comprising adding one or more primaryamine-containing compounds to a culture medium capable of supportinggrowth of microorganisms, wherein said one or more primaryamine-containing compounds are present in an amount effective forneutralization and/or inactivation of any carbapenems that may bepresent in said culture medium.
 2. The method of claim 1, wherein saidculture medium further comprises one or more adsorbents effective forneutralizing and/or inactivating any additional antibiotics present insaid test sample.
 3. The method of claim 1, wherein a test samplesuspected of containing microorganisms is added to said culture mediumand cultured under conditions sufficient for growth of anymicroorganisms in said test sample.
 4. The method of claim 3, whereinsaid primary amine-containing compound is added to the culture medium asa supplement prior to, or concurrently with, said test sample.
 5. Themethod of claim 1, wherein said primary amine comprises a primary amineof the formula R—NH₂ where R is a linear or branched alkyl, aryl,alkaryl, or aralkyl group having between about 1 and about 20 carbonatoms.
 6. The method of claim 4, wherein said primary amine is selectedfrom the group consisting of methylamine, ethanolamine, trisamine,propylamine, 2-aminoheptane, 2-amino-2-methyl-1,3 propanediol,2-amino-2-methyl-1-propanol, n-amylamine, benzylamine,1,4-butanediamine, n-butylamine, cyclohexylamine, ethylamine,ethylenediamine, α-methylbenzylamine, phenethylamine, isopropylamine,butylamine, sec-butylamine, iso-butylamine, andtris(hydroxymethyl)aminomethane.
 7. The method of claim 1, wherein saidprimary amine is an amino acid.
 8. The method of claim 1, wherein saidprimary amine-containing compound is cysteine.
 9. The method of claim 1,wherein said primary amine is immobilized on a solid support and whereinsaid solid support is nitrocellulose, silica, polystyrene,polypropylene, polyvinyl chloride, EVA, glass, carbon, glassy carbon,carbon black, carbon nanotubes or fibrils, platinum, palladium, gold,silver, silver chloride, iridium, rhodium, or a polymeric carrier.
 10. Amethod for the neutralization and/or inactivation of an antimicrobial ina culture medium, the method comprising adding one or more non-thiolcontaining primary amines to said culture medium capable of supportinggrowth of microorganisms, wherein said one or more non-thiol containingprimary amines are present in an amount effective for neutralizationand/or inactivation of any antimicrobials that may be present in saidculture medium.
 11. The method of claim 10, wherein said culture mediumfurther comprises one or more adsorbents effective for neutralizingand/or inactivating any additional antibiotics present in said testsample.
 12. The method of claim 10, wherein a test sample suspected ofcontaining microorganisms is added to said culture medium and culturedunder conditions sufficient for growth of any microorganisms in saidtest sample.
 13. The method of claim 10, wherein said non-thiolcontaining primary amine is added to the culture medium as a supplementprior to, or concurrently with, said test sample.
 14. The method ofclaim 10, wherein said antibiotics comprise one or more β-lactamantibiotics and wherein said β-lactam antibiotics are selected from thegroup consisting of β-lactam antibiotics include, but are not limitedto, penicillins, cephalosporins, monobactams, carbapenems andderivatives thereof.
 15. The method of claim 10, wherein said non-thiolcontaining primary amine is selected from the group consisting ofmethylamine, ethanolamine, trisamine, propylamine, 2-aminoheptane,2-amino-2-methyl-1,3 propanediol, 2-amino-2-methyl-1-propanol,n-amylamine, benzylamine, 1,4-butanediamine, n-butylamine,cyclohexylamine, ethylamine, ethylenediamine, α-methylbenzylamine,phenethylamine, isopropylamine, butylamine, sec-butylamine,iso-butylamine, and tris(hydroxymethyl)aminomethane.
 16. The method ofclaim 10, wherein said primary amine is hydroxylamine.
 17. The method ofclaim 10, wherein said primary amine is immobilized on a solid supportand wherein said solid support is nitrocellulose, silica, polystyrene,polypropylene, polyvinyl chloride, EVA, glass, carbon, glassy carbon,carbon black, carbon nanotubes or fibrils, platinum, palladium, gold,silver, silver chloride, iridium, rhodium, or a polymeric carrier.
 18. Amethod for the diagnosis of an infection caused by a microorganism,comprising the steps of: (a) obtaining a test sample for which thepresence or absence of said microorganism is to be determined, andwherein said test sample may contain one or more carbapenem antibioticswhich may interfere with the growth and/or detection of saidmicroorganism; (b) adding said test sample to a culture medium, saidculture medium comprising at least one primary amine-containing compoundin an amount that is effective for neutralizing, binding or inhibitingsaid carbapenems; and (c) analyzing said culture for the presence ofsaid microorganism, wherein detection of the presence of saidmicroorganism indicates a positive diagnosis for said infection.
 19. Themethod of claim 18, wherein said test sample and culture medium areincubated for a sufficient time and at a sufficient temperature forgrowth of said microorganism prior to step (c).
 20. The method of claim18, wherein said culture medium further comprises one or more adsorbentseffective for neutralizing and/or inactivating any additionalantibiotics present in said test sample.
 21. The method of claim 18,wherein said sample is a clinical sample selected from the groupconsisting of blood, serum, plasma, blood fractions, urine, and salivasamples.
 22. The method of claim 18, wherein said primaryamine-containing compound is added to the culture medium as a supplementprior to, or concurrently with, said test sample.
 23. The method ofclaim 18, wherein said primary amine-containing compound comprises aprimary amine of the formula R—NH₂ where R is a linear or branchedalkyl, aryl, alkaryl, or aralkyl group having between about 1 and about20 carbon atoms.
 24. The method of claim 18, wherein said primaryamine-containing compound is selected from the group consisting ofmethylamine, ethanolamine, trisamine, propylamine, 2-aminoheptane,2-amino-2-methyl-1,3 propanediol, 2-amino-2-methyl-1-propanol,n-amylamine, benzylamine, 1,4-butanediamine, n-butylamine,cyclohexylamine, ethylamine, ethylenediamine, α-methylbenzylamine,phenethylamine, isopropylamine, butylamine, sec-butylamine,iso-butylamine, and tris(hydroxymethyl)aminomethane.
 25. A kit fordetecting microorganisms in a test sample, said kit comprising: (1) asealable specimen container, having an internal chamber comprising aculture medium and in which a test sample may be cultured; and (2) asupplement comprising one or more primary amine-containing compounds.26. The kit of claim 25, wherein said supplement is added to saidspecimen container and said culture medium and wherein said one orprimary amine-containing compounds are present in said culture medium atan amount that is effective for neutralizing, binding or inhibiting anyantimicrobials that may be present in said culture medium.
 27. The kitof claim 26, wherein a test sample is added to said specimen containerand said culture medium and wherein said primary amine is present insaid culture medium in an amount that is effective for neutralizing,binding or inhibiting any antimicrobials that may be present in saidtest sample.
 28. The kit of claim 5, wherein said sealable specimencontainer and said culture medium are sterilized by autoclaving.
 29. Thekit of claim 25, wherein said primary amine-containing compoundcomprises a primary amine of the formula R—NH₂ where R is a linear orbranched alkyl, aryl, alkaryl, or aralkyl group having between about 1and about 20 carbon atoms.
 30. The kit of claim 25, wherein said primaryamine-containing compound is selected from the group consisting ofmethylamine, ethanolamine, trisamine, propylamine, 2-aminoheptane,2-amino-2-methyl-1,3 propanediol, 2-amino-2-methyl-1-propanol,n-amylamine, benzylamine, 1,4-butanediamine, n-butylamine,cyclohexylamine, ethylamine, ethylenediamine, α-methylbenzylamine,phenethylamine, isopropylamine, butylamine, sec-butylamine,iso-butylamine, and tris(hydroxymethyl)aminomethane.